Dynamic changes in genomic 5-hydroxymethyluracil and N6-methyladenine levels in the Drosophila melanogaster life cycle and in response to different temperature conditions

In this study, the level of DNA modifications was investigated in three developmental stages of Drosophila melanogaster (larvae, pupae, imago) and in an in vitro model (Schneider 2 cells). Analysis was carried out using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. Our method made it possible, for the first time, to analyze a broad spectrum of DNA modifications in the three stages of Drosophila. Each stage was characterized by a specific modification pattern, and the levels of these compounds fluctuated throughout the D. melanogaster life cycle. The level of DNA modification was also compared between insects bred at 25 °C (optimal temperature) and at 18 °C, and the groups differed significantly. The profound changes in N6-methyladenine and 5-hydroxymethyluracil levels during the Drosophila life cycle and as a result of breeding temperature changes indicate that these DNA modifications can play important regulatory roles in response to environmental changes and/or biological conditions. Moreover, the supplementation of Schneider 2 cells with 1 mM L-ascorbic acid caused a time-dependent increase in the level of 5-(hydroxymethyl)-2′-deoxyuridine. These data suggest that a certain pool of this compound may arise from the enzymatic activity of the dTET protein.


DNA extraction from Schneider 2 cells
DNA extraction from cells was performed according to a previously published protocol (Starczak et al. 1 ). In brief, a pellet of frozen cells was dispersed in the ice-cold buffer B (Tris-HCl (10 mmol/L), Na2EDTA (5 mmol/L) and deferoxamine mesylate (0.15 mmol/L), pH 8.0). Next 60 µL of RNase A (2 mg/mL, Sigma), 20 µL of RNase T1 (2 U/µL, Sigma) and SDS solution (to a final concentration of 0.5 %) were added, and the mixture was gently mixed using a polypropylene Pasteur pipette. The samples were incubated at 37 °C for 30 minutes. Proteinase K was added to a final concentration of 4 mg/mL and incubated at 37 °C for 1.5 h. The mixture was cooled, transferred to a centrifuge tube with phenol:chloroform:isoamyl alcohol (25:24:1), vortexed vigorously and centrifuged for 15 min at 2800 × g at 4 °C. After the extraction, the aqueous phase was treated with a chloroform:isoamyl alcohol mixture (24:1) and centrifuged under the same conditions. The supernatant was treated with three volumes of cold 96 % (v/v) ethanol to precipitate high molecular weight nucleic acids. The precipitate was removed with a plastic spatula, washed with 70 % (v/v) ethanol and dissolved in 50 µL of the Milli-Q grade deionized water.

Mass-spectrometry profiling of modified nucleotides
The analyses were performed using a method described earlier by Gackowski et al. 2 with some modifications. Briefly speaking, the chromatographic separation was performed with a Waters Acquity 2D-UPLC system with photo-diode array detector for the first dimension chromatography (used for the quantification of unmodified deoxynucleosides) and Xevo TQ-XS tandem quadrupole mass spectrometer (used for the second dimension chromatography and compounds analyzed after the first dimension: 5-hmdC, 5-mdC, 5-formyl-2'-deoxycytidine and 8-oxodG, to assure the better ionization at the higher acetic acid concentration). At-column-dilution technique was used between the first and second dimension to improve the retention at the trap/transfer column. The columns used were as follows: a Waters Cortecs T3 column (150 mm×3 mm, 1.6 µm) with a precolumn at the first dimension, a Waters X-select C18 CSH (100 mm×2.1 mm, 1.7 µm) at the second dimension and Waters X-select C18 CSH (20 mm×3 mm, 3.5 µm) as a trap/transfer column. The chromatographic system operated in a heart-cutting mode, indicating that selected parts of effluent from the first dimension were directed to the trap/transfer column via 6-port valve switching, which served as an "injector" for the second dimension chromatography system. The flow rate at the first dimension was 0.5 mL/min and the injection volume was 2 µL. The separation was performed with a gradient elution for 10 minutes using a mobile phase 0.05 % acetate (A) and acetonitrile (B) (0.7-5 % B for 5 minutes, followed by the column washing with 30 % acetonitrile and re-equilibration with 99 % A for 3.6 minutes). The flow rate at the second dimension was 0.35 mL/min. The separation was performed with a gradient elution for 10 minutes using a mobile phase 0.01 % acetate (A) and methanol (B) (1-50 % B for 4 minutes, isocratic flow of 50 % B for 1.5 minutes, and re-equilibration with 99 % A up to the next injection). All the samples were analyzed in three to five technical replicates the technical mean of which was used for further calculation. Mass spectrometric detection was performed using the Waters Xevo TQ-XS tandem quadrupole mass spectrometer, equipped with an electrospray ionization source. Collision-induced dissociation was obtained using argon 6.0 at 3 x 10-6 bar pressure as the collision gas. Transition patterns for all the analyzed compounds along with specific detector settings were determined using the MassLynx 4.2 Intelli-Start feature in a quantitative mode to assure the best signal-to noise ratio and the resolution of 1 at MS1 and 0.75 at MS2 (Supplementary Table S17 Supplementary Table S17 Transition patterns-specific detector settings and sources of standards for the analysed compounds.

Analysis of L-ascorbic acid levels in cells and culture medium
In order to analyze the concentration of L-AA, both in the culture medium and inside the cells, the previously described ultra-performance liquid chromatography (UPLC) with the UV detection method was used (Modrzejewska et al. 3 ). In brief cells were suspended in 25 µL of cold phosphate-buffered saline (PBS), and 25 µL of precooled 10% (w/v) meta-phosphoric acid aqueous solution were added to obtain L-AA stabilization and the cell membrane perforation. Also 50 µL aliquots of medium samples were treated in the same manner. Then, cells suspension was sonicated for 5 min and incubated on ice for 30 min. Next, the samples were diluted 1:1 with Milli-Q grade deionized water, vortexed and centrifuged at 24 400 × g for 15 min at 4 °C. The supernatants were purified by ultrafiltration using AcroPrep Advance 96-Well Filter Plates 10 K and injected into Waters Acquity UPLC system. The method was validated using the reference material from Chromsystems. The intracellular concentration of ascorbate was calculated under the assumption that mean diameter of S2 cell equals to 9.9±0.33 µm.
The samples were analyzed on Waters Acquity UPLC HSS T3 column (150 mm×2.1 mm, 1.8 µm) with a flow rate 0.25 mL/min and 2 µL injection volume. Ammonium formate (10 mM, pH 3.1) was used as Solvent A and acetonitrile was Solvent B. The following program was used for the ascorbate elution: 0-0.1 min 99 % A, 1 % B, 0.1-2.2 min 97 % A, 2.2-4.0 min -linear gradient to 90 % A, 4.0-4.5 min -90 % A, 4.5-6.0 min -99 % A. The column thermostat was set at 10 °C. The effluent was monitored with a photo-diode array detector at 254 nm, and analyzed with Empower software.
Supplementary Table S18 Flow gradient used for elution during determination of intracellular L-AA concentration in D. melanogaster.

Determination of thymine in acidic hydrolysates of Drosophila extracts
The determination of thymine in homogenates from three developmental stages of D. melanogaster (larvae, pupae, imago) was performed using the method described by Modrzejewska et al. 4 with some modifications. Namely, 20 μL of insect homogenate was incubated at 130 °C for 1 h with 200 μL of 515 μM caffeine in 2 M HCl in a sealed 2 mL glass vial. The cooled sample was completely dried under nitrogen (XcelVap, Biotage AB), dissolved in 50 μL of the Milli-Q grade deionized water and ultrafiltered prior to the injection. A 2 μL aliquot of the sample was chromatographed in duplicate at a flow rate of 0.45 mL/min and 30 °C on CORTECS UPLC T3 1.6 µm (3 x 150 mm) column coupled to Waters Acquity UPLC system with a photo-diode array detector, using two solvents: A -10 mM ammonium formate (pH 3.14) and B -acetonitrile, according to the following elution program: 0-0.1 min, isocratic, 0.1 % B; 0.1-2 min, linear gradient 0.1 %-15 % B; 2-3 min min, linear gradient 15 %-50 % B; 3-3.5 min, isocratic, 50 % B; 3.5-3.51 min, linear gradient, 50 %-0.1 % B. The effluent was monitored with a photo-diode array detector at 254 nm, and analyzed with the Empower software.